The structure of the mitochondrial genome and its role in mitochondrial function are largely unexplored. Mistakes in the regulation of mitochondrial DNA can lead to a number of human diseases that can manifest at any age and in any organ including motor neuropathies, kidney dysfunction, infertility, cardiomyopathy, and neurodegeneration. Generally, the packaging of DNA and its resulting higher-order structure are critically involved in the regulation of gene expression, genome replication, and genome segregation in both eukaryotic and prokaryotic organisms. It is well known that the mitochondrial genome exists as a nucleoprotein complex called a nucleoid, and a single mitochondrion hosts, on average, 5 copies of the genome, resulting in between 100 and 10,000 copies per cell. There is a significant lack of knowledge on how the genome is structured in organello, how this structure differs between cell types, and how this structure is misregulated in disease. Further, it is not known if individual nucleoids within a single mitochondrion have different structures and serve different functions. A critical first step to understanding the structure of the mitochondrial genome is to develop and optimize techniques to characterize the overall three-dimensional architecture of the nucleoid. We propose adapting several powerful, high-resolution technologies, and combining the knowledge gained from each to develop models of the organization of the mitochondrial nucleoid. We will optimize Hi-C to map physically interacting regions of the mitochondrial genome, ATAC-seq and NOMe-seq to determine regions of open and accessible DNA, and ChIP-nexus to develop high-resolution maps of the major nucleoid binding proteins. We will carry these studies out in several distinct human cell lines, which have been shown to have different energy requirements and thus different mitochondrial gene expression. Further, we will adapt this system to differentiating myoblasts in which mitochondrial biogenesis is highly upregulated. Using these powerful techniques, we will determine the role of the major transcription factor and structural protein TFAM in establishing and maintaining genome structure. We will use known disease mutants to understand how the genome structure is disturbed in disease. Finally, we believe that heterogeneity may exist between nucleoids within a mitochondrion. To this end, we will develop methods to fractionate different populations of nucleoids from mitochondria to characterize how their overall architectures differ. In summary, this proposed research will advance our knowledge of mitochondrial genome structure, allowing us to better understand how mitochondrial genes are regulated and how genome replication and copy number are regulated. This knowledge will be invaluable in understanding how mitochondrial mutants lead to disease.